| Neuronal networks in the medulla oblongata generate basic respiratory rhythm in mammals and it is essential for life to keep normal rhythmic respiration.However, little is known about the precise site for respiratory rhythm generation and the mechanisms underlying respiratory rhythmogenesis.It is common in clinical work for opioid-or prostaglandin-induced respiratory depression and many diseases such as central respiratory failure and central respiratory rhythm disturbance,which induce the patient to die.So elucidating the mechanisms underlying respiratory rhythmogenesis clearly is singificant in not only pathophysiology but also preventing and curing respiratory central diseases.In order to study mechanisms underlying respiratory rhythmogenesis conveniently,scholars made the model of medulla oblongata,spinal cord brainstem slices and pre-B(o|¨)tC"island"of neonatal rats.The researchers in the neural science field had paid much attention to the precise site for respiratory rhythm generation and up to now there are some points as below about it:Firstly,professor Zhong-Hai Wu has demonstrated that the medial area of nucleus retrofacialis(mNRF)was the site of respiratory rhythmogenesis in 1986,it was vital to respiratory rhythmogenesis. Sencendly,Smith conformed that the pre-B(o|¨)t complex(PBC)was the site of respiratory rhythmogenesis in 1991.Thirdly,the experimental results of Onimaru showed the rostral ventrolateral medulla(RVLM)was the critical site of respiratory rhythm generation.But in other worker ideas the vicinities of nucleus facialis(VFN) is the important site.The four site statemented as above are all located in the medullary lateral region of abdomen.The anatomical position of them has difference, but it overlapped.The site of basic respiratory rhythmogenesis has been ascertained in the medullary head lateral region of abdomen.There are two hypothesis for the mechanism of respiratory rhythmogenesis,pacemaker neuron hypothesis and neurons network hypothesis.Pacemaker neuron hypothesis is supported by majority of the present findings.We had found Expiratory-Inspiratory phase spanning(E-I PS) neuron characteristic of pacemaker on mNRF in vivo and in vitro in neonate rats.The type of E-I PS neurons begin to discharge before inspiration,burst to initiation inspiration by constant frequency,continue and terminate simultaneously with discharge of inspiration.The type of E-I PS neurons may be pacemaker neurons.There are many neurotransmitters and neuromodulators modulating the respiratory network.As one of the important neuromodulators of catecholamines in the central nervous system(CNS),dopamine(DA)is involved in many physiological and pathophysiological activities such as learning and memory,respiration and blood pressure regulation as well as cocaine craving.The dopamine membrane receptors include five subtypes:D1R-D5R.D1R and D5R activate adenylate cyclase(AC)via Gs protein and increasing the intracellular cAMP level to generate biological effect but D2R-D4R have the opposite effect and decreasing the intracellular cAMP level.As one of the neuromodulators,DA may be involved in the rhythmic discharges of respiration.D1R agonist increased the discharge activities of phrenic nerve and inspiratory neurons by intravenous administration in vivo,meanwhile D1R antagonist had the opposite influence on them.D1R agonists might be therapeutically useful for the treatment of opioid disturbances of breathing without impeding analgesia.Such results show the receptor may be modulate the respiratory rhythm.Activation and suppress D1R would influence the intracellular cAMP level.The stimulation of cAMP production in opossum kidney cells by D1R agonist was dose dependent,and markedly higher levels were observed in the presence of dopamine plus a phosphodiesterase inhibitor,3-isobutyl-1-methylxanthine.The effect of D1R agonist on the opossum kidney cells blocked by a specific D1R antagonist, Sch-23390.Smith found that in cultured embryonic chick motoneurons intracellular cAMP levels increased by 33%following exposure to 100 microM dopamine. Another findings suggested the presence of D1R mediating the cAMP generation system in renal,pulmonary and mesenteric arteries,but SCH-23390 reversed the effect of D1R agonist.As the second messager,cAMP play one important role in cell signal transduction as well as in modulating respiratory rhythmogenesis.Clonidine,which might decrease intracellular cAMP level viaα2-receptors,induced long-lasting depression depression of the respiratory rhythm(C4 and Pre-I activity)in brainstem-spinal cord preparation from newborn rat.The effects of IBMX and Db-cAMP were similar to those of forskolin,they all enhanced the burst rate of isolated Pre-I neurons.cAMP is important in respiratory rhythm generation;this may be due to its regulation of the intrinsic burst generating properties of Pre-I neurons. Opioid-and PGE-evoked respiratory depression was reversed upon elevation of endogenous cAMP levels by stimulating adenylyl cyclase with forskolin,activating dopamine D1R or preventing cAMP breakdown with isobutylmethylxanthine. Increasing the intracellular cAMP level or supressing PKA by a PKA inhibitor affect the respiratory rhythm discharge activities,the cAMP-PKA pathway modulates excitability of inspiratory neurons in the preBotC and,therefore,regulates respiratory rhythm.Moreover,the basal level of endogenous PKA activity appears to be a determinant of resting respiratory frequency.D1R plays an role in regulating respiratory activity in mammals,however,little is known about how this receptor acts to modulate the basic respiratory rhythmgenesis in brainstem-spinal cord preparation from neonatal rat in vitro.Here, by simultaneously recording the activities of biphasic expiratory(BE) neurons/inspiratory(â… )neurons and theâ…«nerve rootlets from brainstem slices in order to solve the questions as below:To explore how D1R affect the respiratory rhythmic discharge activity(RRDA)ofâ…«nerve rootlets;to research the modulating of D1R on BE neurons/I neurons;to study the effect of cAMP on respiratory neurons; to survey how D1R expresses in mNRF"island",the last but important purpose is to explore the underlying mechanisms of D1R regulating the basic respiratory rhythmgenesis.1.The influences of D1R on RRDA ofâ…«nerve rootlets1.1 The effect of different concentration of A68930 on RRDAPerfusing the slices with different concentration A68930,a D1R agonist,the respiratory cycle(RC)and the expiratory time(TE)shortened gradually,Concurrently, integral amplitude(IA)increased gradually.Among the different concentration group of A68930;RC(F=48.663,P＜0.001),TE(F=53.655,P＜0.001)and IA(F=7.760, P=0.002)are statistically significant,respectively.The changes of the three parameters in 5μmol/L group altered most obviously.But in 5μmol/L group the changes of RC,TE and IA at 3 min point were not statistically significant when they were compared with that at 5 min point,the value of P were 0.797,0.809,0.226, respectively.Steady drug response was observed 3～5 min after application.So the suitable concentration of A68930 in our experiments was 5μmol/L.1.2 The effect of different concentration of SCH-23390 on RRDA Perfusing the slices with different concentration SCH-23390,a D1R antagonist, RC(F=70.561,P＜0.001)and TE(F=79.659,P=0.000)extended gradually, Concurrently,TI(F=21.800,P＜0.001)shortened gradually,IA(F=6.617,P=0.004) decreased gradually.Among the different concentration group of SCH-23390;RC (F=70.561,P＜0.001),TE(F=79.659,P=0.000),TI(F=21.800,P＜0.001)and IA (F=6.617,P=0.004)are statistically significant,respectively.The changes of the three parameters in 2μmol/L group are most obviously.But in 2μmol/L group the changes of RC,TI,TE and IA at 3 min point were not statistically significant when they were compared with that at 5 min point,the value of P were 0.874,0.854,0.865,0.238, respectively.Steady drug response was observed 3-5 min after application.So the suitable concentration of SCH-23390 in our experiments was 2μmol/L.1.3 The effect of A68930 and A68930 + SCH-23390 on RRDAAfter perfusing the slices with 5μmol/L A68930,compared to that of control group,RC and TE shortened markedly(P＜0.001),respectively.Concurrently,IA (F=6.617,P=0.004)increased noticeably.Moreover,the effect of A68930 on the respiratory rhythm was partially reversed by additional application of A68930+ SCH-23390.2.The regulating effect of D1R on bisphsic expiratory(BE)neurons/inspiratory(I) neurons in brainstem-spinal cord preparation from newborn rat2.1 D1R modulating the discharge activities of I neuronsAfter perfusing the slices with 5μmol/L A68930,when compared with control, RC and TE of I neurons shortened 20.90%(P=0.000)and 21.91%(P=0.000), respectively.Frequency(Fn)and IA increased 14.84%(P=0.001)and 14.27% (P=0.001),respectively.At the same time the change of TI was not statistically significant(P=0.825).These effects were reversed by subsequent application of D1R antagonist SCH-23390.2.2 D1R affecting the discharge activities of BE neuronsCompard with that of control;RC and TE of BE neurons in A68930 group shortened 20.90%(P=0.000)and 21.91%(P=0.000),respectively;peak frequency(PFn) and IA increased 14.84%(P=0.001)and 14.27%(P=0.001),respectively;at the same time the change of TI was not statistically significant(P=0.844).These effects were reversed by subsequent application of D1R antagonist SCH-23390.3.The role of cAMP in effect of D1R on BE neurons/I neurons in brainstem-spinal cord preparation from neonatal rats3.1 cAMP increasing/decreasing agent acting the discharge activities of BE neurons/I neurons in mNRF3.1.1 cAMP increasing/decreasing agent modulating the discharge activities of I neurons in mNRFFirstly according to the results of previous of us and the literatures,we decided the suitable concentration of IBMX,clonidine,forskolin and Rp-cAMPS were 5,10, 5 and 10μmol/L,repectively.3.1.1.1 IBMX and clonidine acting on the discharge activities of I neuronsAfter 5 min perfusing the slices with 5μmol/L IBMX,RC and TE of IBMX group compared with that of control;RC and TE of I neurons in IBMX group shortened 18.85%(P=0.003)and 20.18%(P=0.003),respectively;TI extended 8.82%(P=0.000);Fn and IA increased 17.16%(P=0.001)and 11.85%(P=0.001), respectively.Expectedly,these effects of IBMX were reversed by subsequent application of clonidine,a cAMP decreasing agent;the parameters above statement were returned to the level of control.3.1.1.2 forskolin and Rp-cAMPS regulating the discharge activities of I neurons Compared with that of control,RC and TE of I neurons in forskolin group shortened 18.81%(P=0.004)and 20.18%(P=0.003),respectively;TI extended 8.34% (P=0.000);Fn and IA increased 18.96%(P=0.001)and 12.36%(P=0.001), respectively.Expectedly,these effects of forskolin on the discharge activities of I neurons were reversed by subsequent application of Rp-cAMPS,an inhibitor of PKA; the parameters above statement were recover to the level of control.3.1.2.1 IBMX and clonidine acting on the discharge activities of BE neuronsAfter 5 min perfusing the slices with IBMX,RC and TE of IBMX group compared with that of control,RC and TE of I neurons in IBMX group all shortened markedly(P=0.000),TI extended significantly(P=0.003),PFn and IA all increased remarkably(P=0.000).Expectedly,these effects of IBMX were reversed by subsequent application of clonidine;the five parameters above mention were returned to the level of control.3.1.2.2 forskolin and Rp-cAMPS modulating the discharge activities of BE neuronsCompared with that of control,RC and TE of BE neurons in forskolin group shortened 31.94%(P=0.000)and 23.53%(P=0.000),respectively;TI extended 10.17%(P=0.012);PFn and IA increased 19.55%(P=0.000)and 30.60%(P=0.000), respectively.Expectedly,these effects of forskolin on the discharge activities of BE neurons were reversed by subsequent application of Rp-cAMPS;the parameters above statement were recover to the level of control.3.2 The role of clonidine and Rp-cAMPS in action of A68930 on BE neurons/I neurons in brainstem-spinal cord preparation from neonatal rats3.2.1 The groups of inspiratory neurons3.2.1.1 Clonidine and clonidine+A68930 regulating the discharge activities of I neuronsAfter 5 min perfusing the slices with clonidine,RC and TE of clonidine group compared with that of control;RC and TE of I neurons in clonidine group extended 21.68%(P=0.001)and 23.45%(P=0.000),respectively;TI shortened significantly (P=0.000);Fn and IA decreased 11.08%(P=0.000)and 12.35%(P=0.002), respectively.Notably,after pretreatment with clonidine,the excitatory effects of A68930 on I neurons were blocked;compared with the five parameters of clonidine group,the changes of RC,TI,TE,IA,Fn were not statistically significant and the values of P were 0.651,0.654,0.639,0.404 and 0.610,respectively.3.2.1.2 Rp-cAMPS and Rp-cAMPS+A68930 modulating the discharge activities of I neuronsCompared with that of control;RC and TE of BE neurons in Rp-cAMPS group extended 22.04%(P=0.000)and 23.81%(P=0.000),respectively;TI shortened 14.29%(P=0.000);at the same time IA and Fn decreasing 13.86%(P=0.000)and 15.85%(P=0.000).Importantly,5 min after pretreatment with Rp-cAMPS,the excitatory effects of A68930 on I neurons were blocked.Compared with the five parameters of Rp-cAMPS group,the changes of RC,TI,TE,IA,Fn were not statistically significant and the values of P were 0.716,0.688,0.704,0.605 and 0.579, respectively.3.2.2 The groups of BE neurons3.2.2.1 Clonidine and clonidine+A68930 regulating the discharge activities of BE neuronsAfter 5 min perfusing the slices with clonidine,RC and TE of clonidine group compared with that of control;RC and TE of I neurons in clonidine group extended 25.93%(P=0.001)and 31.99%(P=0.000),respectively;TI shortened significantly (P=0.005);Fn and IA decreased 10.45%(P=0.001)and 11.37%(P=0.000), respectively.Notably,after pretreatment with clonidine,the excitatory effects of A68930 on I neurons were blocked;compared with the five parameters of clonidine group,the changes of RC,TI,TE,IA,PFn were not statistically significant and the values of P were 0.389,0.945,0.337,0.774 and 0.668,respectively.3.2.2.2 Rp-cAMPS and Rp-cAMPS+A68930 modulating the discharge activities of BE neuronsCompared with that of control;RC and TE of BE neurons in Rp-cAMPS group extended 10.78%(P=0.001)and 13.41%(P=0.000),respectively;TI shortened 8.33% (P=0.035);meantime,IA and Fn decreasing 15.31%(P=0.000)and 17.41% (P=0.000),respectively.Importantly,5 min after pretreatment with Rp-cAMPS,the excitatory effects of A68930 on I neurons were blocked.Compared with the five parameters of Rp-cAMPS group,the changes of RC,TI,TE,IA,PFn were not statistically significant and the values of P were 0.942,0.759,0.881,0.688 and 0.751, respectively.4.Determination of cAMP of neurons in mNRF"island"Determination the concentration of cAMP of neurons in mNRF"island"by ELISA.After incubation the"island"with A68930,the concention of cAMP increased remakably when comparing with that of control.On the contrary,after incubation the"island"with SCH-23390,the concentration of cAMP decreased significantly when comparing with that of control.5.The expression of mRNA of D1R gene in mNRF"island"By real-time PCR we detected the expression of mRNA of D1R gene in mNRF"island".The results suggested that mRNA of D1R gene expressed in mNRF"island"and the quantity of expression was 0.24 times of that of pre-frontal cortex,where the gene expressed plentifully according to the literatures reported.Conclusion1.D1R affected the respiratory rhythmic discharge activity(RRDA)of hypoglossal nerve roots.2.D1R modulated the discharge activities of BE neurons/I neurons,the specific agonist of D1R excited the discharge activities of respiratory neurons and the specific antagonist of D1R had the opposite effect on the neurons. 3.Activating or inhibiting D1R influenced the intracellular level of cAMP of respiratory neurons.4.mRNA of D1R gene expressed in mNRF"island"and the quantity of expression was about 0.24 times of that of pre-frontal cortex.5.Forskolin and IBMX enhanced the discharge activities of BE neurons/I neurons,but clonidine and Rp-cAMPS had the opposite effect on the neurons.6.Clonidine and Rp-cAMPS blocked the excitatory effects of A68930 on the respiratory neurons.7.The intracellular level of cAMP acted to modulating the discharge activities of the neurons.8.The current study indicated that D1R would modulate basic breathing rhythmgenesis via cAMP-dependent mechanisms.
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